bioremediation of colored pollutants-m
TRANSCRIPT
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Author version: Critical Reviews in Microbiology, vol.34; 189-206
Treatment of colored effluents with lignin-degrading enzymes: An emerging
role of marine-derived fungi
Chandralata Raghukumar* Donna D’Souza -Ticlo and Ashutosh Kumar Verma
National Institute of Oceanography,
Council for Scientific and Industrial Research,
Dona Paula, Goa 403 004,
India
* Corresponding author Email: [email protected]
Fax # +91 832 2450606
Running title: Decolorization of colored effluents with marine-derived fungi
Abstract
Some of the industries that discharge highly colored effluents are paper and pulp mills,
textiles and dye-making industries, alcohol distilleries and leather industries. Terrestrial white-rot
basidiomycetous fungi and their lignin-degrading enzymes laccase, manganese-peroxidase and
lignin peroxidases are useful in the treatment of colored industrial effluents and other
xenobiotics. Free mycelia, mycelial pellets, immobilized fungi or their lignin-degrading enzymes
from terrestrial fungi have been reported in treatment of several effluents. Marine obligate or
facultative (marine-derived) fungi may have unique properties but have not been explored
sufficiently for this purpose. This article presents a critical review of bioremediation potential of
such fungi and their lignin-degrading enzymes in comparison with the state-of-the-art in
terrestrial white-rot fungi.
Keywords: Industrial effluents, decolorization, detoxification, laccase, peroxidases, marine
fungi.
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1. Introduction
Paper and pulp mills, molasses based-alcohol distilleries, tanneries, dye-making units and
textiles are some of the major industries that produce and discharge highly colored effluents.
Each of these industrial effluents creates some specific problem besides producing aesthetically
unacceptable intense coloring of soil and water bodies. They block the passage of light to the
lower depths of the aquatic system resulting in cessation of photosynthesis, leading to anaerobic
conditions, which in turn result in the death of aquatic life causing foul smelling toxic waters.
Colored industrial wastewater is usually treated by physico-chemical processes. These
processes include flocculation, flotation, electro flotation, membrane-filtration, ion exchange,
irradiation, precipitation, ozonation and adsorption using activated carbon or biological
adsorption using bacteria, fungi, algae or plant biomass (Robinson et al. 2001; Husain 2006;
Whiteley and Lee 2006 and references therein). Both living and dead cells have been used for
bio-adsorption (Fu and Viraraghavan 2001).
Bioremediation is also possible with naturally occurring (Reddy 1995) or genetically
modified organisms (Chen et al. 1999) for treatment of soil, ground or surface water for general
protection of human health and the environment. This has to be primarily safe and comparatively
less expensive than conventional treatments. Bacteria and fungi along with their products such as
enzymes (Whiteley and Lee 2006) and exopolymeric substances (Liao et al. 2001) aid in
bioremediation. The application of bioremediation has remained limited due to incomplete
understanding of the degradation processes performed by organisms in natural systems and
engineering of suitable systems for the optimum utilization of the organism is required (Pritchard
et al. 1996). Maintaining the optimum catalytic activity of an organism for a long period of time
under controlled conditions for treatment of massive volumes of effluents is a rather difficult
task. Therefore bioremediation has not been as successful as physical and chemical methods
(Pritchard et al. 1996).
A number of biotechnological approaches have been tried for the treatment of colored
effluents and one of the most successful groups of organisms in this context has been the white-
rot basidiomycetous fungi that are capable of extensive degradation of lignin under aerobic
conditions. White-rot fungi are known to play a major role in mineralization of the lignin
polymer to CO2 and H2O in the terrestrial environment. These fungi produce a wide range of
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lignin-degrading enzymes (LDEs), which in turn act on lignin and lignin-analogous compounds.
The LDEs share common features such as broad substrate specificity, high redox potential and
are mostly extracellular in nature. The high redox potential and broad substrate specificity,
increases the range of pollutants the enzyme is capable of degrading. These features combined
with the fact that LDEs are mostly expressed under nutrient deficient conditions (which is
usually the case in the nature) and their ability to oxidize substrates with low solubility have
made them the preferred candidates for bioremediation along with the fungi, responsible for their
production (Reddy 1995).
The process of bioremediation can be monitored by measuring any of the following
factors: (i) by measuring the redox potential, together with pH, temperature, oxygen content and
concentrations of electron acceptor (s) / donor(s) and the breakdown products such as carbon
dioxide or (ii) by measuring chemical oxygen demand (COD) and biological oxygen demand.
Biological oxygen demand (BOD) represents only the organic matter which is capable of being
degraded / oxidized by microbes whereas COD represents all the oxidizable matter, including
organic matter in any particular effluent (Marmagne and Coste 1996). For colored effluents,
bioremediation is measured by estimating the reduction in color units of effluents and percentage
of detoxification achieved besides measuring a few of the above-mentioned parameters.
2. Lignin-degrading enzymes
Lignin-degrading enzymes (LDEs) belong to two classes viz the heme-containing peroxidases
and the copper-containing laccases. A series of redox reactions are initiated by the LDEs, which
degrade the lignin (or lignin-derived pollutants). The LDEs oxidize the aromatic compounds
until the aromatic ring structure is cleaved, which is followed by further degradation with other
enzymes.
Peroxidases are heme-containing enzymes that comprise manganese-dependant
peroxidase (MnP), lignin peroxidase (LiP) and versatile peroxidase (VP). Lignin peroxidase (EC
1.11.1.14) requires H2O2 as the co-substrate as well as the presence of a mediator like veratryl
alcohol to degrade lignin and other phenolic compounds (Fig. 1). Here H2O2 gets reduced to H2O
by gaining an electron from LiP (which itself gets oxidized). The oxidized LiP then returns to its
native reduced state by gaining an electron from veratryl alcohol and oxidizing it to veratryl
aldehyde. Veratryl aldehyde then gets reduced back to veratryl alcohol by gaining an electron
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from lignin or analogous structures such as xenobiotic pollutants. This results in the oxidation of
lignin or the aromatic pollutant (ten Have and Teunissen 2001). MnP (EC 1.11.1.13). This too,
requires H2O2 as its co-substrate and the presence of Mn²+ (naturally present in wood). It catalyses oxidation of Mn²+ to Mn³+, resulting in an Mn³+ chelate-oxalate, which in turn oxidizes
the phenolic substrates. The resulting Mn³+ chelate-oxalate is small enough to diffuse into areas
inaccessible even to the enzyme, as in the case of lignin or analogous structures such as
xenobiotic pollutants buried deep within the soil, which are not necessarily available to the
enzymes (Tortella et al. 2005).
A novel enzyme which can utilize both veratryl alcohol and Mn2+, versatile peroxidase
(EC 1.11.1.16) has been recently described as a new family of ligninolytic peroxidases
(Marti’nez 2002; Marti’nez et al. 2004). The most noteworthy aspect of versatile peroxidase
(VP) is that it combines the substrate-specificity characteristics of LiP, MnP as well as
cytochrome c peroxidase (Du et al. 1992). In this way, it is able to oxidize a variety of (high and
low redox potential) substrates including Mn+2, phenolic and non-phenolic lignin dimers,
veratryl alcohol, dimethoxybenzenes, different types of dyes, substituted phenols and
hydroquinones (Heinfling et al. 1998). It has an Mn-binding site similar to MnP and an exposed
tryptophan residue homologous to that involved in veratryl alcohol oxidation by LiP. It is
suggested that the catalytic properties of the new peroxidase is due to a hybrid molecular
architecture combining different substrate-binding and oxidation sites (Camarero et al. 2000).
Laccase (EC 1.10.3.2) is a benzenediol:oxygen oxidoreductase (a multi-copper enzyme),
present across the kingdoms from bacteria, eg. Azospirillum lipoferum and actinomycetes like
Streptomyces, to fungi to plants and even in insects (Baldrian 2006). This multi-copper oxidase
has the ability to oxidize phenolic compounds. Unlike peroxidases, it does not contain heme as
the cofactor but copper. Neither does it require H2O2 as the co-substrate but rather molecular
oxygen. Laccase often sports a high degree of glycosylation, which confers a degree of self-
resistance to attack by proteases (Yoshitake et al. 1993). The downside however, is that the
redox potential although varying between different laccase isozymes, cannot be compared with
that of the peroxidases, especially LiP. Although laccase does not have the exclusive requirement
for the presence of mediators like veratryl alcohol and Mn+2, their presence increases the
effective range of substrates, which can be degraded by laccase. Laccase, can not only catalyze
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depolymerizing reactions but polymerizing reactions as well. Whilst depolymerization is
obviously useful for the breakdown of pollutants, polymerization can also be useful, even though
larger compounds are created. This is because sequestration is acceptable as a method for
bioremediation. While forming a larger compound does not remove it from the environment, it
can be rendered non-toxic thus negating the need for its removal (Ali and Sreekrishnan 2001).
Based on the enzyme production patterns of white-rot fungi, Hatakka (1994) suggested
three categories of fungi: 1) lignin peroxidase-manganese peroxidase group, 2) manganese
peroxidase-laccase group and 3) lignin peroxidase-laccase group. The most efficient lignin
degraders are able to mineralize lignin to CO2 and belong to the first category of fungi. Only
moderate and very poor mineralization of lignin occurs in the second and third category of fungi
respectively. The terrestrial white-rot fungus Phanerochaete chrysosporium which produces
multiple isozymes of MnP and LiP but mostly no laccase, has been the laboratory model for
physiological and molecular biological studies of LDEs (Fu and Viraraghavan, 2001). Trametes
versicolor producing laccase as the major LDE has been studied widely for industrial application
in biobleaching of paper pulp, treatment of effluents and various other industrial applications
(Wesenberg et al. 2003). However, production of MnP in strains of T. versicolor has also been
demonstrated recently (Snajdr and Baldrian, 2007; Diorio et al. 2008).
3. What and why marine fungi for lignin degradation?
Mangrove plants and seagrasses contain 50 % lignocellulosic material as structural
polymers and are the major contributors of lignocellulose substrate in coastal marine
environment (Benner and Hodson 1985). Mangrove leaves, twigs, wood pieces and seagrasses
fallen into the intertidal zone are colonized by epibiotic bacteria and epi- and endobiotic fungi
(Fig. 2). The term ‘marine-derived’ fungi, is used here since the marine ecosystem comprises of
obligately marine as well as facultative marine fungi. The facultative forms although having
counterparts in the terrestrial ecosystem, have adapted to the marine environment. Obligate and
facultative marine fungi colonizing these substrates produce cell wall-degrading enzymes and are
responsible for the production of dissolved organic carbon (DOC) and particulate organic carbon
(POC) in water (Newell 1996). The resulting DOC is utilized by bacteria for biomass build up
and the microbially colonized POC is utilized as feed by detritus-feeding larvae and other
macroorganisms such as crabs and shrimps (Odum et al. 1979).
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Fungi have been isolated and enumerated from mangrove leaves, seagrasses and salt
marsh grass (Sathe and Raghukumar 1991; Newell 1993; Raghkumar et al. 1995). Several
reports have demonstrated active loss in weight of various timber blocks colonized by marine
wood-degrading fungi (Nilsson et al. 1989; Pointing et al. 1998; Pointing and Hyde 2000;
Bucher et al. 2004). Interestingly, most of these reported fungi belong to ascomycetes and a very
few to basidiomycetes or white-rot fungi. Enumeration of fungi, their succession and
decomposition of mangrove wood is reported from various tropical and subtropical parts of the
world by numerous workers (Vrijmoed and Tan, 1990; Chinnaraj and Untawale 1992;
Vishwakiran et al. 2001). Therefore, fungi growing under such marine conditions are expected
to have adapted to grow under saline (ranging from 10-34 ppt) and alkaline conditions since the
pH of seawater ranges from 7.5 to 8.2. Such LDE-producing fungi should find application in
bioremediation of lignin-based derivatives in colored industrial pollutants such as paper and pulp
mills, tanneries, molasses-based distilleries and textile mills. These effluents are mostly alkaline
and have high salt content (Bartlett 1971) and therefore, marine fungi, facultative or obligate that
grows in the presence of saline and alkaline conditions perhaps are well suited for treatment of
such effluents.
3.1. Lignin-degrading ability of marine fungi
Mineralization of 14C (ring)-labeled synthetic lignin to 14CO2 is considered the acid test
for the lignin-degrading ability of any fungus (Kirk and Farrell 1987). Sutherland et al. (1982)
demonstrated limited mineralization of 14C-labeled maple and spruce lignin to 14CO2 by a
number of marine fungi. Only 5-6% of the labeled lignin was mineralized at the end of 30 days
by these fungi. Phaeospheria spartinicola, an ascomycetous fungus growing on the decaying
leaves of the salt marsh cord grass Spartina alternifolia was shown to degrade lignocellulose and
contribute to dissolved DOC formation (Bergbauer and Newell 1992). After 45 days of
incubation, only 3.3 % of the lignin moiety was mineralized to 14CO2 and 2.7 % solubilized to
DO14C by this fungus. An obligate marine fungus Halosarpheia ratnagiriensis (strain NIOCC
#321) and one facultative marine fungus Sordaria finicola (NIOCC #298) mineralized about 9-
10 % of the U-ring 14C-labeled lignin to 14CO2 within 21 days (Raghukumar et al. 1996). A
basidiomycete, NIOCC #312 isolated from decaying leaves of the sea grass Thalassia hemprichii
on the other hand, mineralized 21 % of the U-ring 14C-labeled lignin to 14CO2 within 21 days
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(Raghukumar et al. 1999). In the same experiment, the lignin-degrading terrestrial fungus
Phanerochaete chrysosporium generally used as a benchmark for lignin-degradation was shown
to mineralize about 21% of the 14C-labeled lignin (DHP) to 14CO2 within 21 days (Raghukumar
et al. 1999). Thus, this marine-derived fungus, NIOCC #312 has been the only one reported
among the marine fungi to match the efficiency of terrestrial white-rot fungi in lignin
mineralization.
3.2. Lignocellulose-degrading enzymes in marine fungi
Lignocellulose-degrading enzymes; cellulase and xylanase have been detected in marine
fungi isolated from salt marsh grass (Gessner 1980). Rohrmann and Molitoris (1992) have also
reported the presence of laccase in addition to the above enzymes in marine fungi isolated from
algae. Marine basidiomycetes and ascomycetes grown in seawater media showed higher laccase
activity than those grown in fresh water media (Rohrmann and Molitoris, 1992). Schaumann et
al. (1986) demonstrated laccase activity in 65 % of marine ascomycete Lulworthia sp. in
substrates like guaiacol, naphthol and benzidine. Subsequently, presence of laccase, cellulase and
xylanase activities in several facultative and obligate marine fungi isolated from mangrove and
seagrass leaves and sediments from mangrove stands were reported (Raghukumar et al. 1994).
About 70% (12 out of 17 fungi screened) of these fungi showed laccase activity and ~ 80 % of
the fungi showed cellulase activity when grown in media prepared with half strength sea water.
Among these, two of the marine ascomycetous fungi Halosarpheia ratnagiriensis (NIOCC #321)
and Sordaria fimicola (NIOCC #298) secreted MnP and laccase in seawater media. Thus, these
two fungi belong to the second category of lignin-degrading fungi, which are classified to
produce MnP and laccase (Hatakka 1994). Pointing et al. (1998; 1999) reported presence of
laccase, cellulase and xylanase in several marine fungi from tropics. Although a thorough list of
marine fungi in tropical America and Africa is available (Kohlmeyer and Kohlmeyer 1979) and
other tropical countries (Kohlmeyer 1984), lignin-degrading activity of these fungi have not been
investigated. On the other hand, large amount of information is available on biologically active
natural product chemistry from marine and marine-derived fungi (Liberra and Lindequist 1995;
Bugni and Ireland 2004) but not on lignin-degrading enzymes. Recently a number of
filamentous fungi have been isolated from hypersaline environment of the Dead Sea (Molitoris et
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al. 2000). They were demonstrated to decolorize several synthetic dyes at various salinities but
no information is available regarding presence of LDE system in these.
Lignin-degrading marine-derived fungi that do not fall into any of the categories
described by Hatakka (1994) have also been reported. The basidiomycetous fungus NIOCC
#312, isolated from decaying seagrass (Thalassia hemprichii) of the Lakshadweep island, India,
produced all three LDEs, also does not conform to any of the above categories (Raghukumar et
al. 1999). However, the major LDEs in this fungus were MnP and LiP whereas laccase was
minimal (Table 1).
Another basidiomycetous fungus NIOCC #2a, isolated from decaying mangrove wood in
Chorao island, Goa, showed laccase positive reaction when grown in LNM prepared with
seawater containing 4 mM guaiacol or 2 mM ABTS (2,2’-azino-bis-(3-ethyl benzothiazoline-6-
sulphonic acid). Production of an intense brown color under and around the fungal colony in
guaiacol-supplemented agar and a deep green color in ABTS-supplemented agar was considered
a positive reaction for the presence of laccase activity (D’Souza et al. 2006)(Table 1). It
produced MnP and LiP in LNM at negligible levels and thus too, does not fall into any of the
reported categories of the lignin-degrading fungi. Several obligate marine fungi have been
reported to produce only laccase (Pointing et al. 1998; Luo et al. 2005). Thus, it appears that
lignin-degrading marine fungi may not be strictly classified in to groups as described by Hatakka
(1994). However, it would be interesting to screen for the presence of all the LDE genes in
marine fungi in general.
The test of adaptation of marine-derived fungi to their environment is growth and
production of degradative enzymes in seawater media. Although, marine fungi showed growth
and LDE activities in agar media (Raghukumar et al. 1994; Pointing et al. 1998) or in liquid
media containing sea water (Raghukumar et al. 1999; Li et al. 2002a; 2002b; D’Souza et al.
2006) laccase and MnP activity of NIOCC #312 and NIOCC #2a, decreased drastically when
seawater was added during enzyme assay (in vitro) studies (unpublished results). Purified laccase
from NIOCC #2a was not inhibited in the presence of NaCl roughly upto 0.3 M, above which it
was reversibly inhibited (unpublished results). Luo et al. (2005) reported that inhibitory effect of
seawater on the laccase activity of two marine fungi tested was reversible. A basidiomycetous
fungus Phlebia sp (strain #MG-60) isolated from mangrove stands was identified as a
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hypersaline-tolerant lignin-degrading fungus (Li et al. 2002a; 2003a) which participated in
biodegradation of sugarcane bagasse, biobleaching of paper pulp and decolorization of dyes (Li
et al. 2002b) in the presence of different concentrations of sea salts. Subsequently these authors
showed production of hypersaline–tolerant MnP in #MG-60, in the presence of sea salt and NaCl
(Li et al. 2003b). Raghukumar et al. (1999) and D’Souza et al. (2006) demonstrated growth and
LDEs production in NIOCC #312 and NIOCC #2a respectively, in media prepared with 50 %
diluted seawater. These two cultures also decolorized several synthetic dyes and industrial
effluents when grown in seawater medium.
Since ascomycetes are more dominant than basidiomycetes in the marine environment
(Kohlmeyer and Kohlmeyer 1979), it is to be expected that ascomycetes would play a major role
as biomass degraders in marine habitats. In vitro production of cellulase and xylanase was
reported among 47 ascomycetes obtained from mangrove stands of tropics (Bucher et al. 2004).
Lignin-degrading enzyme production among these was comparatively less common. Most
isolates were able to cause loss in birch wood mass, when used as substrate during a 24-week
period. Five of these ascomycetous fungi solubilized lignin, with indices of lignin-solubilization
comparable to terrestrial white-rot basidiomycetes. The authors conclude that to a certain extent,
marine ascomycetes in the marine realm play a similar ecological role as that of terrestrial white-
rot fungi (Bucher et al. 2004). Ascomyceteous species were shown to participate in the decay of
dead plant biomass in salt marshes (Lyons et al. 2003).
Recent approach has been to screen for the laccase gene in marine environmental samples
to assess their role in lignin degradation. Analysis of the fungal community in the salt marsh
ecosystem using the diversity of the functional laccase gene indicated high diversity of laccase
sequences in clones from environmental DNA and ascomyceteous fungi isolated from the
decaying blades of Spartina alterniflora (Lyons et al. 2003).
3.3 Effect of nutritional parameters on production of lignin-degrading enzymes
Production of LDEs is affected by several culture conditions such as medium
composition, carbon and nitrogen ratio, pH, temperature and aeration. Production of LiP and
MnP in several terrestrial white-rot fungi was reported in the presence of high carbon and low
nitrogen medium, a condition found in plants. This resulted in the development of a special
culture medium (Tien and Kirk 1988) termed ‘low nitrogen medium’ (LNM). In contrast,
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several white-rot fungi were reported to produce LDEs in the presence of high nitrogen (Kuhad
et al. 1997). Production of LDEs was reported to take place only in shallow undisturbed
stationary cultures (Boominathan and Reddy 1992). Addition of surfactants such as Tween 20 or
Tween 80 to the culture media helped in overcoming this problem and production of these
enzymes in bioreactors and agitated cultures was reported (Gomez-Alarcon et al. 1989). Addition
of manganese to culture medium induced MnP but suppressed LiP production in Phanerochaete
chrysosporium (Boominathan and Reddy 1992). Addition of veratryl alcohol induced both, LiP
and MnP production in several white-rot fungi (Gill and Arora 2003; Boominathan and Reddy
1992). Several natural substrates like wood chips and shavings from soft and hard wood have
been used to induce production of both peroxidases (Niku-Paavola et al. 1990).
With increasing interest in laccase from fungi for bioremdiation applications, efforts have
been made to enhance the laccase titre. Addition of various aromatic compounds analogous to
lignin or lignin derivatives, have induced laccase production (Gianfreda et al. 1999). Nitrogen
source and concentration in the culture medium are known to influence laccase production
(Gianfreda et al. 1999). Addition of copper has been reported to increase laccase production by
several folds (Galhaup and Haltrich, 2001; Galhaup et al. 2002). Recently, novel approaches to
increase laccase production in white-rot fungi by addition of ethidium bromide and a range of
vitamins, amino acids and antibiotics to the culture medium have been reported (Dhawan et al.
2003; Dhawan and Kuhad 2002; Dhawan et al. 2005). To make the fermentation process cost-
effective, optimizing the culture conditions is a prerequisite for large-scale production of these
enzymes. In recent years, several statistical designs collectively under response surface
methodology have been introduced into the fermentation field to replace the “one-factor-at-a-
time” method (Levin et al. 2005). Using these methodologies, production of laccase has been
optimized in several species of white-rot fungi (Levin et al. 2005).
In light of this scenario in terrestrial fungi, marine fungi have received a scant attention.
The marine-derived fungus NIOCC #312 produced both, MnP and laccase in low as well as high
nitrogen medium containing 2.4 mM and 24 mM ammonium tartrate respectively (Raghukumar
et al. 1999). It produced all the three LDEs to varying extents when grown in sea water medium
containing sugarcane bagasse fibres, pine and poplar wood shavings as carbon and nitrogen
source (Table 2). Another marine-derived fungus, NIOCC #2a, produced laccase in low as well
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as high nitrogen medium prepared with seawater of 25 ppt (Table 2). Laccase production was
enhanced by several folds on addition of copper or a combination of copper and guaiacol to 6
day-old culture growing in high nitrogen medium (peptone as the nitrogen source). Several
phenolics and lignin-derivatives also enhanced laccase production in this culture (D’Souza et al.
2006). Glutamic acid and glycine were good sources of nitrogen in the presence of fructose as
carbon source (Table 2) for enhancing laccase production in this fungus (D’Souza-Ticlo et al.
2006).
4. Application of lignin-degrading enzymes
Growing public awareness of the environment is forcing several industrial units to practice
stringent pollution treatment on a top priority. Several strategies including biological approaches
besides physical and chemical methods are devised to restore polluted environments.
Oxidoreductive enzymes play an important role in degradation and transformation of polymeric
substances. The partially degraded or oxidized products can easily be taken up by microbial cells
where they are completely mineralized. Lignin-degrading enzymes are one such group of
oxidoreductive enzymes, which have practical application in bioremediation of polluted
environment (Husain 2006).
4.1. Decolorization of black liquor
The paper and pulp industry release large volumes of intensely colored black-liquors that
contain toxic chlorinated lignin-degradation products. These products include chlorolignins,
chlorophenols and chloroaliphatics (Ali and Sreekrishnan 2001). Besides, these paper mill
effluents are highly alkaline and alter the pH of the soil and water bodies into which they are
discharged. A vast literature is available on the involvement of lignin-degrading fungi from
terrestrial sources in treatment of such effluents (Garg and Modi, 1999). Several white-rot fungi
producing lignin peroxidases are shown to be involved in decolorization of black liquor
(Thompson et al. 2001; Sahoo and Gupta, 2005; Wu et al. 2005) but in a few instances non-
ligninolytic fungi have also been used for this purpose (Nagarathnamma and Bajpai, 1999;
Sumathi and Phatak, 1999). The relative important role of the peroxidases in decolorization of
black liquor has been debated. Frederick et al. (1991) demonstrated negligible decolorization of
bleach plant effluent from paper and pulp mills by a mutant of Phanerochaete chrysosporium
that lacked the ability to produce the peroxidases. A mutant of the same fungus which produced
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only MnPs but not LiPs showed about 80% of the decolorizing activity exhibited by the wild
type, indicating the relatively major role of MnPs in decolorization of this bleach plant effluent.
Very few attempts have been made to tap the potential of marine-derived fungi in
treatment of colored effluents from paper and pulp mills. The marine-derived fungus NIOCC
#312 which produced the peroxidases was better than NIOCC #2a, which produced laccase as
the major LDE, in decolorizing black liquor from paper and pulp mills (Fig. 3). Although the
production of MnP was induced in the presence of black liquor in NIOCC #2a, it did not
decolorize the effluent (D’Souza et al. 2006). Earlier studies demonstrated that marine fungi
Sordaria fimicola (NIOCC #298) and Halosarpheia ratnagiriensis (NIOCC #321), which
produced MnP and laccase, brought about 65-75 % decolorization of bleach plant effluent within
8 days (Raghukumar et al. 1996). These results may indicate that MnP and not laccase plays an
important role in decolorization of black liquor.
The pH of black liquor is always alkaline ranging from 8-11 and most of the lignin-
degrading enzymes show optimum activity between pH 3-6. Therefore, black liquor is usually
acidified prior treatment with these fungi (see references in Wu et al. 2005). These authors have
reported optimum decolorization of black liquor at pH 8-11 using three terrestrial fungi.
Raghukumar et al. (1996) reported decolorization of bleach plant effluent at pH 11 by the isolate
NIOCC #312. These are the only reports demonstrating effective decolorization of black liquor
at alkaline pH.
4.2. Decolorization of molasses spent wash
Molasses spent wash (MSW) is a by-product of sugar mills and alcohol distilleries, where
the starting material is molasses. It contains mostly dark brown colored recalcitrant compounds,
collectively termed as melanoidin, which are formed by the Maillard amino-carbonyl reaction
(Wedzicha and Kaputo 1992). These compounds are toxic to many microorganisms including
those generally involved in wastewater treatment processes (Kitts et al. 1993). Anaerobic
digestion of MSW-containing effluents is one of the treatments followed by distilleries and the
resulting dark brown sludge is used as fertilizer. Dark brown color of these effluents remains a
major problem for the distilleries.
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Color removal from MSW using terrestrial white-rot fungi was shown to be MnP-
dependent in Phanerochaete chrysosporium (Dehorter and Blondeau 1993) and laccase-
dependent in Trametes versicolor (González et al. 2007). The process was sorbose oxidase- and
glucose oxidase-dependent in mitosporic fungi Aspergillus fumigatus (Ohmomo et al. 1987) and
A. oryzae (Ohmomo et al. 1988) and in the basidiomycete Coriolus sp. No. 20 (Watanabe et al.
1982). Raghukumar and Rivonkar (2001) demonstrated MnP-independent decolorization of
MSW by the marine-derived fungus NIOCC #312. About 60% decolorization of MSW was
brought about when added at 50% concentration in seawater medium. Subsequently it was
demonstrated that decolorization was dependent on glucose oxidase levels in the culture medium
(Raghukumar et al. 2004). The activity of MnP and percentage decolorization of MSW by the
isolate NIOCC #312 did not correlate (Fig. 4A) but there was a direct co-relation between
concentration of glucose oxidase and decolorization of MSW (Fig. 4B). It was suggested that
H2O2 produced by glucose oxidase may act as a bleaching agent. In another marine-derived
fungus NIOCC #2a, 80% of color removal was obtained by day 3 (Fig. 3). Glucose oxidase
production in this fungus ranged from 8769 to 23,253 U L-1 but did not correlate with
decolorization of MSW. It was further demonstrated that marine fungi are capable of
decolorizing MSW effectively in the presence of seawater of 15-34 ppt salinity (Raghukumar et
al. 2000; D’Souza et al. 2006). Studies to understand the mechanism of decolorization of MSW
in marine fungi merit further attention.
4.3. Decolorization of synthetic dyes and textile effluents
Textile industries release highly colored effluents containing large amounts of a mixture
of dyes, many of which are resistant to degradation leading to deleterious effects on the aquatic
life (Rodriguez et al. 1999). Exhaustive reviews on decolorization of synthetic dyes (Wong and
Yu 1999; Peralta-Zamora et al. 2003) and dye wastewaters using white-rot fungi and their lignin-
degrading enzymes have appeared (Fu and Viraraghavan 2001; Wesenberg et al. 2003). Textile
effluents besides containing dyes, also have extreme pH values and contains salts, often at very
high ionic strength. Thus, in spite of the highly efficient terrestrial strains reported, marine fungi
may find use in decolorization of industrial effluents with these added factors of salt content and
extreme pH values. The marine-derived culture NIOCC #2a with laccase proved to be more
efficient in the decolorization of textile effluents and synthetic dyes than the culture NIOCC
14
#312 having MnP and LiP activity (Fig. 3). Two textile effluents, textile effluent A (TEA) with
pH of 8.9 and the textile effluent B (TEB) with pH 2.5 were added separately at 10% final
concentration in the fungal cultures grown in seawater medium. Among the synthetic dyes,
Brilliant Green and Congo Red were almost totally decolorized by NIOCC #2a, whereas
Remazol Brilliant Blue R and Poly R-478 were better decolorized by NIOCC #312 than NIOCC
#2a (Fig. 3, Raghukumar et al. 1999; D’Souza et al. 2006).
The role of laccase in terrestrial fungi in decolorization of dyes and dye wastewaters is
undisputed (Wong and Yu 1999; Fu and Viraraghavan 2001; Wesenberg et al. 2003). In the
marine-derived fungus NIOCC #312, decolorization of the dye RBBR directly correlated with
MnP concentration (Fig. 4C). On the other hand, decolorization of the effluents or dyes did not
correlate with laccase concentration in the marine-derived fungus NIOCC #2a (D’Souza et al.
2006). A number of filamentous fungi including a halophilic new species of Gymnoscella
marismortui isolated from the Dead Sea decolorized synthetic dyes belonging to 4 different
groups (Molitoris et al. 2000). However, it is not known whether these fungi produced any of the
lignin-degrading enzymes.
4.4. Degradation of PAHs
Polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion of
fossil fuels. They consist of analogs of benzene having two or more aromatic rings in various
alignments. Most of the low molecular weight PAHs are very toxic and adversely affect aquatic
life. Degradation of PAHs by MnP-producing terrestrial white-rot fungi has been demonstrated
in Irpex lacteus (Baborova et al. 2006), Nematoloma frowardii (Sack et al. 1997), Phanerochaete
chrysosporium (Moen and Hammel 1994) and several other fungi. Laccase-producing white-rot
fungi such as Trametes versicolor (Collins et al. 1996), Pleurotus ostreatus D1 (Pozdnyakova et
al. 2006) and Coriolopsis gallica (Picard et al. 1999) have been implicated in PAHs degradation.
The marine-derived fungi, NIOCC #312 and NIOCC #2a facilitated removal of
phenanthrene, a PAH from the medium by adsorption on the fungal mycelium. Phenantherene
was completely metabolized or transformed into more polar derivatives by NIOCC #312 by day
6 (Raghukumar et al. 2006). Phenanthrene added to the culture NIOCC #2a at 20 ppm
concentration was metabolized to an intermediate compound (Fig. 5A & B). The NMR spectrum
obtained was consistent with earlier data obtained with Phanerochaete chrysosporium, a
15
terrestrial basidiomycete (Sutherland et al. 1991). The mass spectrum of this metabolite showed
a molecular mass of 212 (data not shown). The comparison of the fragmentation pattern of this
peak with the library established its identity to be phenanthrene (trans)-9,10-dihydrodiol
(unpublished results). The fragment ions at m/z 194,181,166,165 and 152 were consistent with
mass spectrum of phenanthrene 9,10-dihydrodiol reported previously (Sutherland et al. 1991). In
both the fungi, majority of phenanthrene removal and further metabolism was by the fungal
mycelia. It was suggested that EPS that form a sheath around the fungal mycelium plays an
active role in the adsorption and degradation of xenobiotic pollutants (Bes et al. 1987). We have
shown the presence of EPS in the isolate NIOCC #312 (Raghukumar et al. 2006) and in NIOCC
#2a (D’Souza et al. 2006). A part of the extracellular LDEs are likely to get trapped in the EPS
surrounding the mycelium, which may further help in degradation of PAHs (Barassa et al. 1998).
5. Effect of pollutants on lignin-degrading enzymes
Several workers have demonstrated the production of LDEs especially laccase, in
terrestrial fungi induced in the presence of aromatic compounds (Mai et al. 2000; Carbajo et al.
2002; Marques et al. 2004). This induction was associated with induced expression of laccase
gene cglcc1 in the presence of tannic acid in the white-rot fungus Coriolopsis gallica and lcc1
and lcc2 in Trametes sp. I-62 (Carbajo et al. 2002; González et al. 2007).
We found that marine fungi were no exception to this. Raghukumar (2000) demonstrated
increased MnP production in three marine fungi, Sordaria fimicola (NIOCC # 298),
Halosarpheia ratnagiriensis (NIOCC #321) and marine-derived basidiomycete (NIOCC #312)
in the presence of bleach plant effluent from a paper and pulp mill. Laccase production in these
fungi was reduced in the presence of bleach plant effluent. In the culture medium containing
either MSW or black liquor, production of MnP was generally enhanced, whereas laccase
production was inhibited in NIOCC #312 (Table 3). In the isolate NIOCC #2a, black liquor and
both the textile effluents in the culture medium induced laccase production whereas, MSW
resulted in decreased laccase production (Table 3). D’ Souza et al. (2006) demonstrated that
textile dye effluent, black liquor and MSW enhanced laccase production in the same fungus.
However, the media used in these two different studies differed in their nitrogen source.
Subsequently, it was proven that laccase production in the presence of effluents was strongly
influenced by the nitrogen source (D’ Souza et al. 2006a).
16
Addition of phenanthrene (12 ppm) increased fungal biomass production in both NIOCC
#312 (Raghukumar et al. 2006) and in NIOCC #2a. However in the presence of phenanthrene,
maximum biomass was attained earlier in NIOCC #2a than in NIOCC # 312 (Fig. 6A & 6B).
6. Methods of treatment of effluents
Several methods of treatment of industrial effluents with fungi for decolorization have been
reported (Thompson et al. 2001). Whole cultures, mycelial pellets, immobilized fungi or their
enzymes, free enzymes and biofilm (Wu et al. 2005) are some examples of such treatments.
6.1. Treatment of effluents with whole versus immobilized cultures
Most of the experiments for decolorization of various effluents and xenobiotics with
terrestrial basidiomycetes are carried out by addition of effluents to pre-grown, shallow, static
cultures having substantial fungal biomass (Reddy 1995). Care should be taken not to disturb the
fungal mat, as this affects LDE production. The efficiency of the fungal mat decreases on
repeated usage. In nature, most of these fungi grow in solid-state conditions, in the near absence
of free water. Such cultures when immobilized on a variety of solid supports have been shown to
decolorize various effluents (Ohmomo et al. 1987; Shin et al. 2002; Šušla et al. 2007). Zhang et
al. (1999) used immobilized a white-rot fungus strain F29 to decolorize Orange II, a toxic dye.
Wu et al. (2005) used biofilm of white-rot fungi grown on a porous plastic rings for
decolorization of paper mill effluent.
The marine-derived fungus NIOCC #312 when immobilized in polyurethane foam (PUF)
cubes, decolorized MSW by 70% by day five and most of the high molecular weight compounds
were removed by this treatment. The same batch of immobilized fungus could be reused up to
four cycles for treatment of MSW. The immobilized fungus remained viable for a minimum of
75 days (Raghukumar et al. 2004). When NIOCC # 2a, was immobilized in PUF, it was able to
decolorize MSW to varying degrees and also reduce COD levels by 50% within 72 h
(unpublished results). The porous nature of PUF increases the diffusion of oxygen, resulting in
increased production of LDEs as well as enhanced activity. Since many of the white-rot fungi
produce LDEs under oxygen-saturation conditions (Boominathan and Reddy, 1992). Thus
immobilized marine fungi offer an alternative technology for an effective environmental
bioremediation.
17
6.2. Treatment of effluents with crude/purified enzyme and immobilized enzymes
Several studies with terrestrial white-rot fungi showed involvement of either mycelial-
bound lignin-degrading enzymes or H2O2 generating mechanism for degradation of lignin and
decolorization of several effluents (Wesenberg et al. 2003; Svabodová et al. 2008). It has been
shown by immunochemical methods that sites of H2O2 production are located in the periplasmic
space of the fungal mycelium in P. chrysosporium (Forney et al. 1982). By using an immuno-
cytochemical technique Garcia et al. (1987) demonstrated that LiPs are located very close to the
cell membrane in Phanerochaete chrysosporium and other white-rot fungi. Further, washed
pellets were shown to retain a part of the lignin-degrading enzyme activity (Kurek and Odier
1990). Earlier studies on lignin-degradation therefore were concentrated using whole cultures of
white-rot fungi showing lignin-peroxidases activity. As reports about laccase-producing white-
rot fungi increased, lignin-degradation work and decolorization applications with partially
purified or crude laccase gained importance (Wong and Yu 1999; Rodriguez et al. 1999; Kokol
et al. 2007; Šušla et al. 2007). Laccase-mediated degradation of PAHs has also been widely
reported (Pozdnyakova et al. 2006 and references therein).
Decolorization of various effluents by culture supernatant collected from liquid culture
was possible with the marine-derived fungus NIOCC #2a (Table 4) whereas, the same by
NIOCC #312 was not effective. It is possible that the mycelial-bound lignin-degrading enzymes
or the H2O2-generating mechanism in NIOCC #312 is responsible for decolorization of various
effluents. This isolate produces more peroxidases than laccase and the former require constant
supply of H2O2, which is not possible practically while using mycelia-free culture supernatants.
Immobilized laccases have been extensively used for decolorization of textile effluents
and synthetic dyes (Abadulla et al. 2000; Peralta-Zamora et al. 2003). Various supports such as
alumina particles, chemically modified silica, amberlite and glass-ceramic have been used for
this purpose (Abadulla et al. 2000; Peralta-Zamora et al. 2003). It has been demonstrated that
treatment of azo dyes with free laccase enzyme results in darkening of the solution due to
coupling of the degraded products with the unreacted dyes (Zille et al. 2005). Using immobilized
enzyme would avoid these coupling reactions. This knowledge gained with terrestrial fungus,
Trametes villosa should be considered while using laccases from marine-derived fungi for any
bioremediation processes.
18
6.3. Treatment of effluents with exopolymeric substances (EPS) produced by white-rot
fungi
Color removal by biosorption is an alternative to the economically disadvantageous
physical and chemical methods of treatment (Namasivayam et al. 1996). Color removal by
adsorption is an alternative option. Biological adsorbants include plant, fungal and bacterial
biomass, either live or dead (Robinson et al. 2001). Bioadsorption potential of microbial
exopolymeric substance is well known (Wingender et al. 1999). Basidiomycetous fungi are
reported to produce large amount of EPS (Smith et al. 2002; Maziero et al. 1999). These
polymeric substances form a sheath around the fungal hyphae and may be water soluble or
insoluble forms. The ligninolytic fungus Phanerochaete chrysosporium also produces
polysaccharide sheath and dissolution of this sheath by addition of glucanase inhibited lignin
degradation (Bes et al. 1987). This suggests an active involvement of EPS in lignin degradation
process.
The participation of fungal EPS in degradation of xenobiotics has been assumed but not
yet investigated. D’Souza et al. (2006) reported for the first time its use in decolorization of
colored industrial effluents. Extracellular polymeric substance was produced by the marine-
derived fungi NIOCC #2a and NIOCC #312. Freeze-dried EPS of NIOCC #2a was effective in
decolorizing various industrial effluents (Table 4) but that of NIOCC #312 was not effective for
this purpose. The EPS produced by a marine cyanobacterium Cyanothece sp. was reported to
remove dyes from textile effluents by gelation under alkaline conditions (Shah et al. 1999). EPS
are highly charged and thus absorb water and become gel-like (Whiteley and Lee, 2006). EPS
produced by basidiomycetes function as a supporting network in which some of the excreted
enzymes get trapped (Ruel and Joseleau, 1991). Biofilm prepared with EPS for treatment of
effluent needs to be considered for the future bioremediation processes. Lignin-degrading
enzymes immobilized in fungal EPS is another possible strategy for bioremediation purpose. As
EPS production is reported to be NaCl-dependent in cyanobacteria (Philippis & Vincenzini,
1998), marine fungi should be screened for EPS production.
7. Detoxification of effluents
It has been reported that effluents from paper and pulp mills and textile dye waste waters
are toxic and mutagenic (Reddy 1995). Laccases are shown to render phenolic compounds in
19
effluents, less toxic via degradation or polymerization reactions or by cross-coupling of pollutant
phenols with naturally occurring phenols (Abadulla et al. 2000). Toxicity of several textile dyes,
including azo compounds, was reduced by treatment with laccase from Trametes hirsuta
(Abadulla et al. 2000). Eight white-rot fungi grown in green olives reduced phenolic content by
nearly 70-75 % but phytotoxicity was not reduced (Aggelis et al. 2002). All of these fungi
produced laccase and some of them produced MnP. Rhizomucor pusillus strain RM7, a
mucoralean fungus and a white-rot fungus Coriolus versicolor were shown to detoxify bleach
plant effluent (Driessel and Christov 2001).
Molasses spent wash treated with the marine-derived fungus NIOCC #312 was detoxified
as measured by serum sorbitol dehydrogenase (SSDH) assay (Raghukumar et al. 2004).
Increased levels of SSDH are indicative of chemically induced liver damage in fish (Dixon et al.
1987). Accordingly, there was a drastic reduction in SSDH activity in fungus-treated MSW
(Table 5). Toxicity test was also carried out by Comet assay, a technique to detect DNA damage
in individual cells (Singh et al. 1988). In the presence of untreated MSW, 85% of the hepatic
cells of an estuarine fish Oreochromis mosambicus showed damaged nuclei as comets, whereas,
in the presence of fungus-treated MSW only 9 % of the cells showed presence of comets
indicating a near total removal of toxic components after the fungal treatment (Table 5). There
was a 30% reduction in benzo(�)pyrene, one of the toxic polycyclic aromatic hydrocarbons in
MSW on treatment with the fungus (Table 5), which might be one of the toxic components
present in MSW. A decrease in phenolics and PAHs might be responsible for cumulative
reduction in toxicity of the fungus-treated MSW (Raghukumar et al. 2004). Reduction in COD as
an indicator of detoxification of the effluents has been shown in several white-rot fungi (Table
6). Molasses-based pharmaceutical effluent in the presence of 1 % glucose when added to
NIOCC #2a and NIOCC #312 cultures, showed a 57 and 67 % reduction in COD after 72 h
respectively. The total phenolics were reduced by 20 and 35% respectively in the same treatment
(unpublished results). Thus, besides decolorization, detoxification of wastewaters is an important
parameter to be monitored.
8. Role of mediators in treatment of effluents
Lignin-degradation by white-rot fungi that produce only laccase led to the discovery of low
molecular weight enzyme mediators. These laccase-mediator systems (LMS) involves use of low
20
molecular weight compounds that are oxidized by the enzyme to stable radicals which in turn act
as redox mediators and oxidize other compounds that are not substrates of laccase. Lack of
correlation between laccase activity and degradation of xenobiotic compounds further supports
the role of LMS (Johannes and Majcherczyk 2000). These mediators can either be natural
compounds produced by fungi or plants or synthetic compounds such as, ABTS, 1-
hydroxybenzothiazole (HBT), violuric acid (VIO) and N-hydroxyacetanilide (NHA) (Camarero
et al. 2005). Degradation products of lignocellulose such as acetosyringone, p-coumaric acid,
syringaldehyde, vanillin, can also act as mediators. A metabolite, 3-hydroxyanthranilic acid
produced by the white-rot fungus Pycnoporus cinnabarinus was shown to act as a mediator in
degradation of lignin (Eggert et al. 1996). Natural mediators have been extracted from black
liquor of eucalyptus-based kraft pulping (Camarero et al. 2007). The redox potential of laccase
alone is not high enough to break C-H aliphatic bonds. In the presence of a redox mediator,
oxidation of such bonds becomes feasible. The enzyme oxidizes the mediator, which can diffuse
away from the enzyme and oxidize a substrate. The reduced mediator is ready for the next cycle
(Wells et al. 2006). Laccase-mediator system has found applications in paper pulp delignification
(Camarero et al. 2007), degradation of PAHs (Johannes and Majcherczyk 2000) and industrial
dyes (Camarero et al. 2005).
Preliminary results with the marine-derived fungus NIOCC #2a showed that treatment of
sugarcane baggase with laccase brought about reduction in lignin content, as determined by
kappa number. Lignin content was further reduced in the presence of ABTS, a mediator
(unpublished results). Role of mediators in laccases from marine fungi has remained a totally
ignored subject. As marine-derived fungi are a vast source of biological compounds, search for
the presence of natural mediator compounds in them is an exciting new field.
9. Conclusion and future perspectives
Microbial decolorization and degradation of colored effluents is a cost-effective and
promising green technology for treatment of such effluents. Reports of white-rot fungi that show
lignin-degrading ability in saline conditions are very few. Industrial effluents are mostly alkaline
and rich in carbonates, chlorides and sulfates. In light of this, the marine fungi discussed here
hold good promise for the application of bioremediation of colored effluents under saline
conditions. The marine-derived fungi NIOCC #312 with MnP and LiP and NIOCC #2a with
21
laccase as the major lignin-degrading enzyme were equally effective in decolorization of various
effluents and degradation of PAHs under marine conditions. Marine-derived fungi are often
more effective than terrestrial fungi in treatment of various colored effluents since they are better
adapted to perform under such extreme conditions (Table 6).
Isolation of fungi from marine environment using media prepared with sea water of
different salinities incorporated with lignin model compounds (Poly R 478) or laccase substrates,
guaiacol or ABTS is recommended for obtaining selective lignin-degrading fungi. Growth and
enzyme production of such cultures under saline conditions is to be maintained by growing them
in seawater. Media with low nitrogen and high carbon content is preferable for culturing these
fungi.
Development of EPS-based bioremediation with marine fungi needs concerted efforts of
basic and applied research. Dependency of EPS production on the concentration of NaCl in
cyanobacteria is well known (Philippis and Vincenzini 1998). Similarly, production of EPS in
obligate and facultative marine fungi in the presence of NaCl needs to be studied in detail to
increase the production of EPS in such fungi to bring out a biotechnologically-developed product
in the field of bioremediation. Search for hyper-production of EPS in marine fungi is a
challenging field.
Screening of marine-derived fungi as a source of new naturally occurring mediators for
enhancing laccase production require to be carried out systematically on a large scale.
Understanding the mechanism of degradation and detoxification of industrial effluents by marine
lignin-degrading fungi are some of the key areas for future research.
Most of the LDEs show optimum activity around 30-35oC and at pH of 3-4. Industrial
effluents generally have alkaline pH and temperatures above ambient. Therefore, fungi from the
marine environment with LDE activity at alkaline pH preferably around 40oC should be
selectively isolated for bioremediation purposes. Recombinant enzymes with these properties or
protein engineering of the enzymes should be considered to achieve this goal. Increasing
efficiency of these enzymes for electron transfer should be aimed at, by producing hybrid
enzymes. Effect of carbon and nitrogen sources and pH of the effluent on the fungus and / or
enzyme used for bioremediation should be studied in detail. The efficacy of the isolates in
treating effluents from common effluent treatment (CET) plant which contain mixtures of
22
effluents from various small-sized mills should also be assessed. Laccase can act alone or in the
presence of low molecular weight mediators as well as in the absence of the fungal biomass. On
the other hand, LiP and MnP require an H2O2-generating system from the fungal mycelium and
thus they require presence of the live fungal biomass for lignin-degradation. Immobilized
peroxidase-producing fungi may be tested for this purpose. A laccase-hyperproducing strain
from marine environment using inexpensive growth medium would be a suitable alternative. Co-
immobilization of all the three LDEs or fungal isolates that produce them could be a promising
technology for treatment of colored effluents.
Acknowledgements
Ms D’Souza-Ticlo acknowledges the senior research fellowship from Council for
Scientific and Industrial Research (CSIR), New Delhi. Mr.Verma is grateful to Department of
Biotechnology, New Delhi for a junior research fellowship. The senior author thanks CSIR for
financial support. This is NIO’s contribution No.
23
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1
Table 1. Comparison of the lignin-degrading enzyme production in low nitrogen medium
(LNM) by the two marine-derived fungi, NIOCC #312 and NIOCC #2a.
NIOCC#312
Days
3 5 7 9 11 13 15
MnP(U L-1)
0
47
112
298
338
276
118
LiP(U L-1)
8
12
29
52
15
0.1
0
Laccase(U L-1)
0.8
0.24
30.5
1.4
2.3
0.6
0.6
Days
NIOCC#2a
3 5 7 9 11 13 15
MnP(U L-1)
1.5
80
82
79
84
105
93
LiP(U L-1)
0.3
0.5
0
0
0
0.4
0.6
Laccase(U L-1)
571
8425
5097
6125
8946
12613
7415
2
Table 2. Production of lignin-degrading enzymes (U L-1) by the two marine-derived fungi
grown in seawater media. Maximum activity recorded in each media is shown.
Culture Media MnP LiP Laccase
NIOCC #312
Low nitrogen medium (LNM)*
654 52 30
High nitrogen medium (HNM)*
50 0 20
Sugarcane bagasse*
450 20 15
Pine shavings*
110 4 9
Poplar shavings*
110 0 8
NIOCC #2a
LNM with Ammonium tartrate as N source
40 0 3428
HNM (peptone as N source)** 13 1 921
HNM + Cu**
37 0 42,933
LNM with KNO3 as N source***
0 26 16,687
LNM with glycine as N source***
12 2 34,659
LNM with glutamic acid as N source***
0 17 47,567
LNM with beef extract as N source***
25 25 10,346
LNM with corn steep liquor as N source***
46 26 12,973
*Source data: Raghukumar et al. 1999; ** source data: D’Souza et al. 2006a;
*** source data: D’Souza et al. 2006b
3
Table 3. Effect of effluents on lignin-degrading enzyme production in NIOCC #312 and
NIOCC #2a. All the effluents were added to 10% final concentration in the culture
medium.
LDETreatment Enzyme Activity (U L-1) in NIOCC #312
Day 5 Day 7 Day 9 Day 11 Day 13 Day 15
MnP
Without effluent 47 112 298 338 276 118 With MSW 51 78 263 226 225 142
With BL 16 255 331 483 66 13 With TEA 104 0 0 54 0 0 With TEB 52 0 0 0 0 0
Laccase
Without effluent 0 0 0 0 0 0 With MSW 0 0 16 56 56 40
With BL 1 1 0 1 0 0 With TEA 104 0 0 54 0 0 With TEB 52 0 0 0 0 0
Enzyme Activity (U L-1) in NIOCC #2a*
MnP
Without effluent 80 82 79 84 105 93 With MSW 0 22 69 78 193 79
With BL 197 513 516 683 530 367 With TEA 86 53 94 0 0 0 With TEB 8 14 8 48 13 0
Laccase
Without effluent 8425 5097 6125 8946 12613 7415 With MSW 566 629 644 1266 720 704
With BL 197 513 516 683 530 367 With TEA 5603 3348 3278 3195 4279 2900 With TEB 1728 799 412 676 1549 848
* Source data: D’Souza et al. 2006a. MSW=molasses spent wash; BL=black liquor from
paper and pulp mill; TEA= textile effluent A; TEB=textile effluent B.
4
Table 4. Decolorization of effluents by fungus-free extracellular culture supernatant and
extracellular polymeric substance (EPS) produced by the fungus NIOCC #2a (D’Souza et
al. 2006a)
Culture supernatant (500 �l) with 9 U ml-1 laccase activity was incubated with an equal
volume of diluted effluent at pH 6.0 and 60oC. Freeze-dried EPS (10 mg) of the fungus
was incubated with effluents at 60oC. Decolorization of both was measured at the
absorbance maxima specific to the effluents. The percentage decolorization was
calculated based on the initial readings. All the values are mean of 2 replicates.
Effluents (10%)
(%) Decolorization by culture supernatant
(%) Decolorization by EPS
6 h 12 h 12 h 24 h
Textile Effluent A 9
11
11
100
Textile Effluent B
14
22
35
100
Molasses Spent Wash
34
33
12
100
Black Liquor
71
59
41
100
5
Table 5. Detoxification of molasses spent wash by NIOCC #312 tested by
various toxicity assays (Raghukumar et al. 2004).
Toxicity Test Untreated MSW Treated MSW
Serum sorbitol dehydrogenase
122 U ml-1
2.6 U ml-1
Comet assay (damaged nuclei)
85 %
9 %
Benzo (�)pyrene
3.8 �g ml-1
1.2 �g ml-1
6
Table 6. Comparison of bioremediation potential of terrestrial white-rot fungi with that of marine-derived fungi
NIOCC #312 and NIOCC #2a.
Effluent Fungus %
Decolorization % Phenol Reduction
% COD Reduction
Enzyme (s) involved
Technique Reference
Alcohol Distillary
Waste (3%)
Pycnoporuscoccineus
50 – 55 (9 days)
70 – 80 (9 days)
193 (9 days)
MnP Immobilised on
Polyurethane foam
Charattanamankorn et al. 2005
Pulp and Paper Mill Effluent
Consortium containing: 1) Merulius
(Syn. Phlebia) aureus. 2) An
unidentified basidiomycete
3) Fusrium Sambucinum
79 (4 days)
--- 89 (4 days)
--- Static Liquid Culture
Malaviya & Rathore 2006
Textile Effluent
Phanerochaetesordida
90 (48 h)
--- --- MnP Static Liquid Culture
Harazono & Nakamura 2005
Olive Mill Wastewater
(20% & 50%)
Phanerochaetechrysosporium
93 (15 days)
90 (15 days)
74 (15 days)
MnP, LiP & Laccase
Static Liquid Culture
Kissi et al. 2001
7
Pulp Mill Effluent
Trametesversicolor
90 (7 days)
--- 69 (7 days)
--- Mycellial Pellets
Meghna et al. 1995
Green Olive Wastewater
Pleurotusostreatus
49 (30 days)
76 (30 days)
12 (30 days)
MnP, MnIP, Laccase
Static Liquid Culture
Aggelis et al. 2002
Olive Mill Effluent
Penicillium spp. 85 – 90 (20 days)
55 – 60 (20 days)
60 – 70 (20 days)
--- Static Liquid Culture
Robles et al. 2000
Paper Mill Effluent
Trametesversicolor
--- 70 (3 h)
--- Laccase with
mediator
Static Liquid Culture
Minussi et al. 2007
MSW Coriolusversicolor
53 (10 days)
--- --- --- Static Liquid Culture
Fitz Gibbon et al 1998
Sugar Refinery
Waste Water
Phanerochaetechrysosporium
55 (3 days)
63 (3 days)
48 (3 days)
--- Immobilised on PUF and
Scouring Web
Guimaraes et al. 2005
MSW (6.25% v/v)
Coriolusversicolor
71.5 (10 days)
90 (10 days)
--- --- Shake Flask Technique
Kumar et al. 1998
Bleach Plant Effluent
Rhizopus oryzae 92 – 95 (24 h)
--- 50 (24 h)
--- Immobilised on PUF
Nagarathnamma et al. 1999
Nitrocellulose Industry Effluent
Aspergillus sp. Lentinus edodes
83 – 95 (24-72 h)
56 – 79 24-72 h)
--- --- Shake Flask Technique
Souza et al. 2005
8
MSW (10%) (NIOCC #312) 75 (3 days)
50 (8 days)
50 (8 days)
--- Static Liquid Culture
Raghukumar & Rivonkar 2001
MSW (10%) (NIOCC #312) 78 – 80 (7 days)
--- --- Glucose Oxidase
Immobilised on PUF
Raghukumar et al. 2004
TEA (10 %) (NIOCC #312) 18 (3 days)
50 ( 3 days)
72 (3 days)
- Static Liquid Culture
In this review
TEB (10 %) (NIOCC #312) 37 (3 days)
22 (3 days)
79 (3 days)
- Static Liquid Culture
In this review
Bleach Plant Effluent
(2000 Color Units)
(NIOCC #312) 80 (8 days)
- - MnP Static Liquid Culture
Raghukumar et al. 1996
BL (10%) . (NIOCC #2a) 40 (72 h)
14 (72 h)
50 (72 h)
- Static Liquid Culture
D’Souza et al. 2006
MSW (10%) (NIOCC #2a) 71 (48 h)
20 (48 h)
50 (48 h)
laccase Static Liquid Culture
D’Souza et al. 2006
TEA (10%) (NIOCC #2a) 64 (6 days)
87 (6 days)
82 (6 days)
laccase Static Liquid Culture
D’Souza et al. 2006
TEB (10%) (NIOCC #2a) 92 (3 days)
69 (3 days)
85 (3 days)
laccase Static Liquid Culture
D’Souza et al. 2006
1
Legends to the Figures
Fig. 1. Schematic diagram illustrating the role and activity of various extracellular lignin-
degrading enzymes produced by fungi (Modified from Field et al. 1993). MnP =
manganese-dependent peroxidase; LiP = lignin peroxidase; VP = versatile
peroxidase
Fig. 2. Schematic diagram illustrating the process of degradation of plant material in the
marine habitat.
Fig. 3. Comparison of the efficiency of the two marine-derived fungi, NIOCC #312 and
NIOCC #2a. The dyes and pollutants were added to the 6-day old cultures and %
decolorization was estimated after 3 days. AB= Azure B; BG= Brilliant Green;
CR= Congo Red; Poly-B= Poly-B 411; Poly-R= Poly-R 478; CV= Crystal Violet;
RBBR= Remazol Brilliant Blue R; BL= Black Liquor; MSW= Molasses spent
Wash; TEA= Textile effluent A; TEB= Textile effluent B. Standard deviation was
< 5%.
Fig. 4. A) No correlation observed between MnP production and decolorization of MSW
in NIOCC #312; B) Correlation observed between glucose oxidase production
and decolorization of MSW in NIOCC #312; C) Correlation observed between
MnP production and decolorization of Remazol Brilliant Blue R dye by NIOCC #
312.
Fig. 5. A) A 300-MHz Proton NMR spectra of the standard phenanthrene; B)
intermediate degradation product, phenanthrene (trans)-9,10-dihydrodiol
extracted from the fungal biomass (NIOCC #2a) on day 6. The numbers on
peaks correspond to the numbers shown in the chemical structures in the insets.
The insets in (A) and (B) show the structure of phenanthrene and phenanthrene
(trans)-9,10-dihydrodiol respectively.
Fig. 6. Effect of phenanthrene on biomass production in A) NIOCC #312 and; B) NIOCC
#2a.
Present inimmediatevicinity
Synt
hesiz
ed de
no
vo
Mediator( reduced state )
Mediator( oxidized state )
Lignin / pollutants Lignin / pollutantsLow molecular weightbreakdown products
H2O( reduced )
H2O2 ( oxidized )
Gluconic acid
Glyoxal / Glucose oxidase
CO2
FUNGAL HYPHA
VP
Aer
obic
con
ditio
ns
Several oxido - reduction steps Several oxido - reduction steps
( + ) mediators( -
) m
edia
tors
Taken up by the fungal
hyph
ae &
min
eral
ized
to
Laccase
Fig.1
LiPMnP
Mangrove leaves & wood /Algae / Seagrass
ShreddersLarge pieces of detritus
Cell w
all - degrading enzymes
Bacteria ( epifilm )
Colonized by fungi
& bacteria
Dissolved Organic Carbon (DOC)
Biomass build upof free - living bacteria
Fungi ( epi - & endobiotic )
Fine pieces of detritus; Particulate Organic Carbon ( POC )
POC - enriched with microbial biomass( Feed for detritivores, eg. prawn larvae )
Leaching
Leaching
Fig. 2
0
20
40
60
80
100
A B BG CR CV Poly-B Poly- R RBBR BL MSW TEA TEB
Dyes / pollutants
NIOCC #312NIOCC #2a
Fig. 3
% D
ecol
oriz
atio
n
r2 = 0.2825
0
20
40
60
80
100
0 200 400 600MnP (U L-1)
% D
ecol
oriz
atio
n of
MSW
A
r2 = 0.9915
0
20
40
60
80
100
0 200 400 600
Glucose Oxidase (U L-1)
% D
ecol
oriz
atio
n of
MSW
B
r 2 = 0.8981
0
20
40
60
80
100
0 200 400 600
MnP (U L-1)
% D
ecol
oriz
atio
n of
RB
BR C
Fig.4
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 ppm
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 ppm
A
B
Fig. 5
1
23
45
6
78 9
10
H OHOH
H
5 4 81
9, 10
63 7, 2
54 8 1
7 2
9, 103 6
0
20
40
60
80
100
120
without phenanthrene
with phenanthrene
A
0
20
40
60
80
100
120
0 3 6 9 12 15
Time (Days)
B
Fig.6
Fung
al b
iom
ass (
mg,
dry
wei
ght)